IgE is a member of the immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity and the familiar sinus inflammation suffered on a widespread basis. IgE is secreted by, and expressed on the surface of, B-cells. IgE binds to B-cells (and monocytes, eosinophils and platelets) through its Fc region to a low affinity IgE receptor (Fc.sub..epsilon. RII). Upon exposure of a mammal to an allergen, B-cells synthesize IgE that binds the allergen. This IgE in turn is released into the circulation by the B-cells where it is bound by B-cells (through the Fc.sub..epsilon. RII) and by mast cells and basophils through the so-called high affinity receptor (Fc.sub..epsilon. RI) found on the surface of the mast cells and basophils. Such mast cells and basophils are thereby sensitized for allergen. The next exposure to the allergen cross-links the Fc.sub..epsilon. RI on these cells and thus activates their release of histamine and other factors which are responsible for clinical hypersensitivity and anaphylaxis.
It is generally understood that Fc.sub..epsilon. RI and Fc.sub..epsilon. RII bind to recognition sites in the IgE constant (Fc) domain. The IgE recognition sites for the two receptors have been poorly defined, despite considerable effort in the past directed to the problem.
Over the past decade several studies have been undertaken to determine which portions of the human IgE molecule are involved in binding to human Fc.sub..epsilon. RI and Fc.sub..epsilon. RII and which portions of the rodent IgE molecules are involved in binding to rodent Fc.sub..epsilon. RI and Fc.sub..epsilon. RII. Essentially three approaches have been tried. First, peptides corresponding to specific portions of IgE sequence have been used as either competitive inhibitors of IgE-receptor binding or to elicit anti-IgE antibodies which would block IgE-receptor interaction. Second, mutations in IgE have been partially explored in efforts to identify the binding site. Third, chimeric molecules have been constructed in attempts to characterize the binding site. These three approaches are discussed below.
For purposes of consistency, all numbering of immunoglobulin amino acid residues, including the amino acid numbering of peptides corresponding to specific portions of IgE, mutant IgE molecules and chimeric IgE molecules, that appears herein is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. 1987). Thus, any numbering system for immunoglobulins or fragments thereof appearing in the references discussed below is ignored and such immunoglobulins or fragments are renumbered herein according to the numbering system of Kabat et al.
In a study using the first approach discussed above, i.e., the IgE peptide approach, Nakamura et al., EP 0263655 published Apr. 13, 1988, found that peptides corresponding to human IgE residues 360-389, 457-523, 464-499, 516-521, 360-380, 376-388, 455-475, 464-472, 497-516, 513-526, 515-521, and 516-530 administered by injection into the skin of rats reduced the size of skin lesions caused by antigen challenge at the treated sites.
Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 (1989) found that a peptide corresponding to IgE residues 329-409 blocked in vivo sensitization of human basophil granulocytes with human IgE antibodies. Further studies indicated that residues 395-409 were not essential for binding of the 329-409 peptide to Fc.sub..epsilon. RI (Helm et al., Proc. Natl. Acad Sci., 86:9465-9469 (1989)).
Vercelli et al., Nature, 338:649-651 (1989) used recombinant IgE peptides as well as anti-Fc.sub..epsilon. monoclonal antibodies to investigate the B-cell (Fc.sub..epsilon. RII) binding site of human IgE. They concluded that the Fc.sub..epsilon. RII binding site is in Fc.sub..epsilon. 3 near K399-V402.
Burt et al., Eur. J. Immun., 17:437-440 (1987) investigated seven peptides corresponding to amino acid sequences located within various surface regions of the C.sub.H 3 and C.sub.H 4 domains of rat IgE for competition against rat IgE in binding to rat mast cells. Their most active peptide, p129, was 1000-fold less active than IgE. p129 is homologous to human sequence 439-453 which includes loop EF. Another of their peptides, p130, homologous to residues 396-418 in the human Fc.sub..epsilon. 3 domain, which includes the CD loop, had no activity.
Robertson et al., Molec. Immun., 25:103-113 (1988) assessed murine IgE binding by sequence-directed antibodies induced by several synthetic peptides. They concluded that the sequence defined by their .epsilon.-peptide-4 (homologous to human IgE residues 446-460 spanning the EF loop), was not significantly involved in receptor binding while the sequence defined by their E-peptide-3 (homologous to human IgE residues 387-401), was likely to be proximal to the IgE-receptor recognition site. Thus, the murine anti-IgE binding study of Robertson et al., Molec. Immun., 25:103-113 (1988) contradicted the Burt et al., Eur. J. Immun., 17:437-440 (1987) data implicating the EF loop in rat IgE binding.
Nio et al., Peptide Chemistry, 203-208 (1990) evaluated numerous peptides with respect to their ability to inhibit histamine release by human basophils in vitro. Only one peptide (peptide 2, Table 1), exhibited specific inhibition; this peptide encompassed residues 376-388. However, a larger peptide which incorporated this sequence (peptide 3, Table 1), had no inhibitory activity.
In a study using the second approach discussed above, i.e., mutations in mouse IgE to characterize the Fc.sub..epsilon. RI-binding site on mouse IgE, Schwarzbaum et al., Eur. J. Immun., 19:1015-1023 (1989) (supra) found that a point mutant P404H (P442H by the numbering system used herein) had 2-fold reduced affinity for Fc.sub..epsilon. RI on rat basophilic leukemia (RBL) cells, but the interpretation of this finding is controversial (Weetall et al., J. Immunol., 145:3849-3854 (1990)).
Lastly, in the third approach discussed above, chimeric molecules have been constructed in attempts to identify the Fc.sub..epsilon. RI-binding site on IgE. Human IgE does not bind to the murine receptor (Kulczycki Jr., et al., J. Exp. Med., 139:600-616 (1974)) while rodent IgE binds to the human receptor with a reduced affinity (Conrad, et al., J. Immun., 130:327-333 (1983)); human IgG1 does not bind to IgE receptors (Weetall et al., J. Immun., 145:3849-3854 (1990)). Based on these observations, several groups have constructed human-murine chimeras or human IgE-IgG chimeras. Weetall et al., J. Immun., 145:3849-3854 (1990) made a series of human IgG1-murine IgE chimeras and concluded that the Fc.sub..epsilon. 2 and Fc.sub..epsilon. 3 domains are involved in binding murine Fc.sub..epsilon. RI while the Fc.sub..epsilon. 4 domain is unlikely to be involved in binding to murine Fc.sub..epsilon. RI (but may possibly be involved in binding to Fc.sub..epsilon. RII). However, these conclusions are uncertain since they rest primarily on lack of binding by chimeras and three of five chimeras lacked some interchain disulfide bonds.
Nissim et al., EMBO J., 10:101-107 (1991) constructed a series of human-murine IgE chimeras and measured binding to RBL-2H3 rat mast cells and concluded that the portion of mouse IgE which binds with high affinity to the specialized Fc.epsilon. receptor on RBL cells could be assigned to Fc.sub..epsilon. 3. In further studies with human-murine IgE chimeras, Nissim et al., J. Immunol., 150: 1365-1374 (1993) reported that although the introduction of the entire mouse Fc.sub..epsilon. 3 domain into human IgE enabled the chimera to bind mouse Fc.sub..epsilon. RI, the swapping of only 16 or 26 amino acids from the N-terminus of mouse Fc.sub..epsilon. 3 for the homologous residues in human IgE (residues 361-377 or 361-388) was insufficient to endow the chimera with mouse Fc.sub..epsilon. RI-binding ability. Moreover, Nissim et al. (J. Immunol.) found that substitution of 26 amino acids from the N-terminus and 47 amino acids at the C-terminus of mouse Fc.sub..epsilon. 3 for the homologous residues in human IgE (residues 361-388 and 438-506) failed to confer mouse Fc.sub..epsilon. RI-binding ability and also reduced human Fc.sub..epsilon. RI-binding ability.
The results reported by these authors (e.g. Helm et al. and Burt et al.) are contradictory as to what part of IgE binds to Fc.sub..epsilon. RI in rodents or humans. However, the instant invention precisely identifies the domains of human IgE which are involved in the binding of human IgE to human Fc.sub..epsilon. RI receptor.
It is an object of this invention to identify antagonists of human IgE which are capable of inhibiting allergic responses.
It is another object to provide novel compounds for use in the assay of human Fc.sub..epsilon. RI receptor and for use as immunogens or for selecting anti-human IgE antibodies.